Cytotoxic drug therapy

ABSTRACT

A compound comprises a target cell-specific portion, such as an antibody specific to tumour cell antigens, and an inactivating portion, such as an enzyme, capable of converting a substance which in its native state is able to inhibit the effect of a cytotoxic agent into a substance which has less effect against said cytotoxic agent. The prolonged action of a cytotoxic agent at tumour sites is therefore possible whilst protecting normal tissues from the effects of the cytotoxic agent.

This invention relates to potentially cytotoxic agents which may betargeted to selected cells, and is particularly concerned with theaction of agents used in the treatment of cancer.

Most forms of cancer tend to disseminate in the body at an early stageand the ultimate aim of cancer therapy is to achieve elimination ofcancers, preferably without incurring serious toxic effects on hostsystems. Combinations of cytotoxic agents have proved curative in asmall range of relatively uncommon cancers, but single agents andcombinations of them have failed to achieve major therapeutic benefitsin most patients with the common cancers of lung, breast, colon, rectum,pancreas, prostate etc.

Cytotoxic agents can only be given by intermittent dose schedulesbecause of their effects on normal tissues in which cell renewal isactive such as haemopoietic tissues and epithelia of the alimentarytract. The rest period between treatments which is necessary to allowrecovery of these normal tissues from the effects of the cytotoxicsubstances tends to be of much greater duration than the period ofadministration of the cytotoxic agents.

Substances involved in cell division are the commonest targets forcytotoxic agents and amongst these are substances involved in thesynthesis of nucleotides, the basic components of DNA and RNA. Theenzymes ribonucleotide reductase, dihydrofolate reductase and thymidinesynthetase are typical targets. The enzyme dihydrofolate reductase actson a dietary factor, folic acid, to produce the active co-enzyme5,10-methenyltetrahydrofolate. The co-enzyme is required for one carbontransfer in various syntheses including that of pyrimidines required forDNA synthesis. The widely used drug methotrexate (2,4-diamino-N¹⁰-methylpteroylglutamic acid) acts by binding strongly to dihydrofolatereductase preventing regeneration of active tetrahydrofolate and thusinterrupting DNA synthesis and leading to death of cells entering Sphase of the cell cycle in which DNA is duplicated. Methotrexate isgenerally available, for example from Cyanamid Inc.

The drug trimetrexate (NSC 352122;2,4-diamino-5-methyl-6-[3,4,5-trimethoxyanilino methyl]quinazoline) alsoacts by binding to dihydrofolate reductase but whereas methotrexateenters cells via the folate receptors, trimetrexate enters byalternative mechanism(s). The synthesis of trimetrexate is disclosed byBaker (1967) in Design of site-directed irreversible enzyme inhibitors,Wiley, New York, and by Elslager et al (1974) Lectures in heterocyclicchemistry, Vol. 2, pp 97/5-133 (Castle & Townsend, ed), Hetero Corp,Oren, Utah. Trimetrexate is generally available from US Biosciences, OneTower Bridge, 100 Front Street, Suite 400, West Conshohocken, Pa. 19428,USA.

Methotrexate resembles natural folates in having a terminal glutamicacid moiety which can be cleaved by carboxypeptidase G2, whereastrimetrexate is not susceptible to the action of this enzyme (Bagshawe(1985) Clinical Radiol. 36, 545-551 ). We have previously reported thatthe action of trimetrexate on colonic cancer cells in vitro can beenhanced by the addition to the culture medium of a folate degradingenzyme carboxypeptidase G2 (Searle et al (1990) Biochemical Pharmacol.39, 1787-1791. We have also shown that this enzyme retains activity whenconjugated to antibodies or antibody fragments (Searle et al (1988)Bact. J. Cancer 53, 377-384).

The biological effect of both methotrexate and trimetrexate can bereversed by administering an end product of the reaction they block, orby a more readily available analogue known as folinic acid [5-formyltetrahydrofolic acid]. Folinic acid is widely available, for example asLeucovorin from Cyanamid Inc, but also from Wellcome Inc, andFarmitalia. If folinic acid is given in sufficient dosage concurrentlywith methotrexate or trimetrexate their actions are blocked. It has beenfound useful in the treatment of some cancers to use folinic acid inconjunction with methotrexate in carefully timed and dose controlledsequences. The methotrexate-folinic acid combination can improve thetherapeutic ratio compared with methotrexate alone for certain cancersand is commonly known as `rescue` therapy. It appears to depend on theability of folinic acid to rescue normal clonogenic cells more readilythan some cancer cells. One example is the successful use ofmethotrexate and folinic acid in the treatment of some trophoblastictumours (Bagshawe et al (1989) Brit. J. Ob. & Gynaecol.). However thisapproach has proved useful in only a limited range of cancers and itseems likely that the time x concentration of folinic acid which isnecessary to protect normal cells also protects some cancer cells fromthe action of the anti-folate.

Moreover the use of folinic acid in this way still necessitatesintermittent administration of the methotrexate (MTX), whereas it wouldbe advantageous to give the MTX more continuously over a prolongedperiod since it has been shown that the duration of action ofanti-folates determines the degree of cytotoxicity achieved. Similarconsiderations apply to other cytotoxic drugs.

One aspect of the present invention provides a compound comprising atarget cell-specific portion and an inactivating portion capable ofconverting a substance which, in its native state, is able to inhibitthe effect of a cytotoxic agent into a substance which has less effectagainst said cytotoxic agent.

The inactivating portion may be directly or indirectly inactivating.

By "directly inactivating" we mean that the portion itself is able toinactivate the said substance, for example by binding to it or byconverting it into an inactive form.

By "indirectly inactivating" we mean that delivery of the portion to thetarget cell results in inactivation of the cytotoxic agent. For example,the portion may be a nucleic acid, either DNA or RNA, that encodes apolypeptide that is able to inactivate the said substance, for exampleby binding to it or by converting it into an inactive form.

The said polypeptide may be expressed intracellularly, may be expressedon the cell surface, or may be secreted from the cell. By the term"polypeptide" we include proteins and glycoproteins.

Preferably, the inactivating portion is an enzymatically active portion.

Substances which "inhibit" the effect of a cytotoxic agent are thosewhich diminish to a useful extent the ability of the cytotoxic agent todestroy target cells. Preferably, the said ability is reduced tosubstantially zero. Similarly, the inactivating portion will reduce suchinhibition to a useful extent and will preferably reduce it tosubstantially zero.

The entity which is recognised by the target cell-specific portion maybe any suitable entity which is expressed by tumour cells,virally-infected cells, pathogenic microorganisms, cells introduced aspart of gene therapy or normal cells of the body which one wishes todestroy for a particular reason. The entity should preferably be presentor accessible to the targeting portion in significantly greaterconcentrations in or on cells which are to be destroyed than in anynormal tissues of the host that cannot be functionally replaced by othertherapeutic means. Use of a target expressed by a cancer cell would notbe precluded, for example, by its equal or greater expression on anendocrine tissue or organ. In a life-saving situation the organ could besacrificed provided its function was either not essential to life, forexample in the case of the testes, or could be supplied by hormonereplacement therapy. Such considerations would apply, for instance, tothe thyroid gland, parathyroids, adrenal cortex and ovaries.

The entity which is recognised will often be an antigen.Tumour-associated antigens, when they are expressed on the cell membraneor secreted into tumour extra-cellular fluid, lend themselves to therole of targets for antibodies.

The term "tumour" is to be understood as referring to all forms ofneoplastic cell growth, including tumours of the lung, liver, bloodcells (leukaemias), skin, pancreas, colon, prostate, uterus or breast.

The antigen-specific portion may be an entire antibody (usually, forconvenience and specificity, a monoclonal antibody), a part or partsthereof (for example an Fab fragment or F(ab')₂) or a synthetic antibodyor part thereof. A conjugate comprising only part of an antibody may beadvantageous by virtue of optimizing the rate of clearance from theblood and may be less likely to undergo non-specific binding due to theFc part. Suitable monoclonal antibodies to selected antigens may beprepared by known techniques, for example those disclosed in "MonoclonalAntibodies: A manual of techniques", H. Zola (CRC Press, 1988) and in"Monoclonal Hybridoma Antibodies: Techniques and Applications", J. G. R.Hurrell (CRC Press, 1982). All references mentioned in thisspecification are incorporated herein by reference. Bispecificantibodies may be prepared by cell fusion, by reassociation ofmonovalent fragments or by chemical cross-linking of whole antibodies,with one part of the resulting bispecific antibody being directed to thecell-specific antigen and the other to the enzyme. The bispecificantibody can be administered bound to the enzyme or it can beadministered first, followed by the enzyme. It is preferred that thebispecific antibodies are administered first, and after localization tothe tumour cells, the enzyme is administered to be captured by thetumour localized antibody. Methods for preparing bispecific antibodiesare disclosed in Corvalan et al (1987) Cancer Immunol. Immunother. 24,127-132 and 133-137 and 138-143, and Gillsland et al (1988) Proc. Natl.Acad. Sci. USA 85, 7719-7723.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition, a fact first recognised byearly protease digestion experiments. Further confirmation was found by"humanisation" of rodent antibodies. Variable domains of rodent originmay be fused to constant domains of human origin such that the resultantantibody retains the antigenic specificity of the rodent parentedantibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By "ScFv molecules" we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Effector functions of whole antibodies, such as complementbinding, are removed. Fab, Fv, ScFv and dab antibody fragments can allbe expressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of the said fragments.

Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" wemean that the said antibodies and F(ab')₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dab fragments aremonovalent, having only one antigen combining sites. Fragmentation ofintact immunoglobulins to produce F(ab')₂ fragments is disclosed byHarwood et al (1985) Eur. J. Cancer Clin. Oncol. 21, 1515-1522.

IgG class antibodies are preferred.

Alternatively, the entity which is recognised may or may not beantigenic but can be recognised and selectively bound to in some otherway. For example, it may be a characteristic cell surface receptor suchas the receptor for melanocyte-stimulating hormone (MSH) which isexpressed in high numbers in melanoma cells. The cell-specific portionmay then be a compound or part thereof which specifically binds to theentity in a non-immune sense, for example as a substrate or analoguethereof for a cell-surface enzyme or as a messenger.

The virally directed enzyme-pro-drug therapy (VDEPT) approach has beendisclosed for the selective killing of neoplastic cells using thetranscriptional differences between normal and neoplastic cells toselectively drive expression of enzymes capable of converting a pro-druginto a cytotoxic drug (Huber et al (1991)Proc. Natl. Acad. Sci. USA 88,8039-8043).

Thus, by analogy, transcriptional differences between normal andneoplastic cells may be used to selectively drive the expression of anenzyme capable of inactivating the inhibitor substances disclosedherein. It is preferred that the enzyme is secreted by the tumour cell,thus allowing free access to the inhibitor substance to be inactivated.

A difference in transcription between cells may be associated withtissue-specific promoters, or may be due to changes in activator orrepressor molecules in the neoplastic state. Thus in one example,liver-associated albumin transcriptional regulatory sequences may beuseful to drive the expression of inhibitor-inactivating proteins,including enzymes, in the treatment of patients with hepatocellularcarcinoma. More transcriptional differences between normal andneoplastic cells are being discovered all the time, and it is believedthat many of these differences may be exploited in the methods of thepresent invention.

Recombinant, replication-defective retroviruses which are suitable fordelivering the genetic constructs (ie promoter plus gene encodinginhibitor-inactivating protein) to target cells have been disclosed(Huber et al (1991) Proc. Natl. Acad. Sci. USA 88, 8039-8043).

Thus, a virus or other nucleoprotein particle, or liposome may provide atarget cell-specific portion suitable for delivering the indirectlyinactivating portion to the said cell.

The inhibitor-inactivating protein may be an enzyme capable ofmetabolising the said inhibitor to an inactive form, or it may be aprotein capable of binding the said inhibitor and hence inactivating it.

Considerable work has already been carried out on antibodies andfragments thereof to tumour-associated antigens and antibodies orantibody fragments directed at carcinoembryonic antigen (CEA) andantibodies or their fragments directed at human chorionic gonadotrophin(hCG) can be conjugated to carboxypeptidase G2 and the resultingconjugate retains both antigen binding and catalytic function. Followingintravenous injection of these conjugates they localise selectively intumours expressing CEA or hCG respectively. Other antibodies are knownto localise in tumours expressing the corresponding antigen. Suchtumours may be primary and metastatic colorectal cancer (CEA) andchoriocarcinoma (hCG) in human patients or other forms of cancer.Although such antibody-enzyme conjugates may also localise in somenormal tissues expressing the respective antigens, antigen expression ismore diffuse in normal tissues. Such antibody-enzyme conjugates may bebound to cell membranes via their respective antigens or trapped byantigen secreted into the interstitial space between cells.

Examples of tumour-associated, immune cell-associated and infectionreagent-related antigens are given in Table 1.

                  TABLE 1    ______________________________________    Antigen    Antibody     Existing Uses    ______________________________________    1. Tumour Associated Antigens    Carcino-embryonic               {C46 (Amersham)                            Imaging & Therapy of colon    Antigen    {85A12 (Unipath)                            /rectum tumours.    Placental Alkaline               H17E2 (ICRF, Imaging & Therapy of    Phosphatase               Travers & Bodmer)                            testicular and ovarian cancers.    Pan Carcinoma               NR-LU-10 (NeoRx                            Imaging & Therapy of               Corporation) various carcinomas incl. small                            cell lung cancer.    Polymorphic               HMFG1 (Taylor-                            Imaging & Therapy of    Epithelial Mucin               Papadimitriou,                            ovarian cancer, pleural    (Human milk fat               ICRF)        effusions.    globule)    β-human Chorionic               W14          Targeting of enzyme (CPG2)    Gonadotropin            to human xenograft    choriocarcinoma         in nude mice (Searle et al                            (1981) Br.J. Cancer 44,                            137-144).    A carbohydrate on               L6 (IgG2a)1  Targeting of alkaline    Human Carcinomas        phosphatase (Senter et al                            (1988) Proc. Natl. Acad. Sci.                            USA 85, 4842-4846    CD20 Antigen               1F5 (IgG2a)2 Targeting of alkaline    on B Lymphoma           phosphatase (Senter et al    (normal                 (1988) Proc. Natl. Acad. Sci.    and neoplastic)         85, 4842-4846    Other antigens include alphafoetoprotein, Ca-125 and prostate    specific antigen.    2. Immune Cell Antigens    Pan T Lymphocyte               OKT-3 (Ortho)                            As anti-rejection therapy    Surface Antigen         for kidney transplants.    (CD3)    B-lymphocyte               RFB4 (Janossy,                            Immunotoxin therapy of B    Surface Antigen               Royal Free   cell lymphoma.    (CD22)     Hospital)    Pan T lymphocyte               H65 (Bodmer, Immunotoxin treatment of    Surface Antigen               Knowles ICRF,                            Acute Graft versus Host    (CD5)      Licensed to Xoma                            Disease, Rheumatoid               Corp., USA)  Arthritis.    3. Infectious Agent-Related Antigens    Mumps virus-               Anti-mumps   Antibody conjugated to    related    polyclonal   Diphtheria toxin for               antibody     treatment of mumps.    Hepatitis B               Anti HBs Ag  Immunotoxin against    Surface Antigen         Hepatoma.    ______________________________________     1 Hellstrom et al (1986) Cancer Res. 46, 3917-3923     2 Clarke et al (1985) Proc. Natl. Acad. Sci. 82, 1766-1770

Before the administration of a conjugate with an antibody directed at atumour associated antigen it may be advantageous to augment theexpression of that antigen at tumour sites since this may increase theamount of conjugate retained at tumour sites. Several agents have beenidentified as able to increase antibody uptake including tumour necrosisfactor (TNF) (Forsyth et al (1988) Cancer Res. 48, 3607-3612) andinterferons (Borden (1988) J. Nat. Cancer Inst. 80, 148-149).

Thus it is preferred that the expression of the tumour cell antigen isenhanced using any one or more of the reagents prior to or during theadministration of the compound of the invention.

Uptake of an antibody, antibody fragment or any uptake thereof may alsobe modified by vasoactive agents through alterations in tumour bloodflow or altering capillary permeability. Such agents include histamineand interleukin-2 (Hennigan et al (1991) Brit. J. Cancer 64, 872-874),flavone acetic acid (Bibby et al (1989) J. Natl. Cancer Institute 81,216-219) but other agents may be used to alter tumour blood flow orcapillary permeability so as to favour increased retention of enzyme attumour sites or inhibit penetration of tumour sites by the protectingmetabolite.

Thus it is further preferred that tumour blood flow is altered using oneor more of the reagents prior to or during the administration of thecompounds of the invention.

The substance which in its native state is able to inhibit the effect ofa cytotoxic agent may be any sufficiently non-toxic substance which maybe converted into a substance which has less effect on said cytotoxicagent. A suitable compound is folinic acid. Folinic acid reverses thebiological effect of the cytotoxic agent trimetrexate, for example,which acts on the enzyme dihydrofolate reductase. Folinic acid isdeglutamated and rendered inactive against trimetrexate by the enzymecarboxypeptidase G2 and other deglutamating enzymes.

The same principle may be applied to other anti-cytotoxic agentsubstances. For example, thymidine blocks the effect of a cytotoxicagent, such as CB3717 and ICI D1694 (Jodrell et al 1991, BJC 64, 833-8;Jones et al (1986) J. Med. Chem. 29,468-472), which acts on the enzymethymidylate synthetase. Hence a thymidine degrading enzyme (such asdihydrothymine dehydrogenase, Shiotani & Weber 1981 J. Biol. Chem. 256,219-224) or thymidine kinase (Shiotani et al (1989) Cancer Res. 49,1090-1094) may be used as the inactivating portion of the compound ofthe invention to render the thymidine ineffective against the cytotoxicagent.

Similar considerations relate to other agents which interfere with thenormal processes of nucleotide incorporation into DNA or RNA since theseare potentially reversible by the normal metabolite which in turn can bedegraded by an appropriate enzyme targeted to tumour sites.

For instance, it has been shown that the cytotoxic effects of the widelyused cytotoxic 5-fhorouracil (available from Roche Products Inc) can beat least partly attenuated by uridine (Groeningen et al (1989) J. Natl.Cancer Inst. 81, 157-162). It follows that conjugation of an antitumourantibody with a uridine degrading enzyme can be used in conjunction with5-fluorouracil and uridine. Such a combination would be particularlyrelevant in colorectal and breast carcinoma for which 5-fluorouracil isone of the most effective cytotoxic agents. Such a combination of agentsmay be further combined with folinic acid which augments thecytotoxicity of 5-fluorouracil or additionally with thymidine and athymidine inactivating enzyme.

The inactivating portion of the compound will be chosen by reference tothe anti-cytotoxic agent substance.

Enzymes other than carboxypeptidase G2 and its equivalents can be used.They should be specific for the targeted metabolite but may be of humanor non-human origin.

It may not be necessary to use a conventional enzyme. Antibodies withcatalytic capacity have been developed (Tramontano et al Science 234,1566-1570) and are known as `abzymes`. These have the potentialadvantage of being able to be humanized to reduce their immunogenicity.

Enzymes derived from human lymphocytes and able to degrade thymidinehave been disclosed. (Schiotani et al (1989) Cancer Res. 49, 1090-1094).A dihydrothymine dehydrogenase and thymidine kinase can be used in thesystem of the type herein disclosed for use in conjunction withinhibitors of thymidine synthetase.

Thymidine degrading and phosphorylating enzymes can be used as anadditional element in anti-folate therapy as herein disclosed byblocking the thymidine salvage pathway. They can also be used inconjunction with uridine catalysing enzymes used with the cytotoxic drug5-fluorouracil.

The bacterial enzymes carboxypeptidase G1 and G2 (CPG1 and CPG2) degradefolates including methotrexate by cleavage of the terminal glutamicacid. The actions of the two enzymes are thought to be the same. Thefollowing description of preferred aspects of the invention refers toCPG2 but is equally applicable to CPG1 and to any other enzymes actingon the same substrates, and to abzymes acting on the same substrates.

The isolation, purification and some of the properties ofcarboxypeptidase G2 from Pseudomonas sp. strain RS-16 have beendisclosed by Sherwood et al (1984) Eur. J. Biochem. 148, 447-453. Thecloning of the gene encoding the said carboxypeptidase G2, itsnucleotide sequence and its expression in E. coli have been disclosed byMinton et al (1984) Gene 31, 31-38 and Minton et al (1983) J. Bacteriol.156, 1222-1227. CP2G2 is available from the Division of Biotechnology,Centre for Applied Microbiological Research, Porton Down, Salisbury, UK.Carboxypeptidase G1 (CPG1) is disclosed by Chabner et al (1972) CancerRes. 32, 2114-2119.

It is likely that the inactivating portion of the compound, when it isan enzymatically active portion, will be enzymatically active inisolation from the cell-specific portion but it is necessary only for itto be enzymatically active when (a) it is in combination with thecell-specific portion and (b) the compound is attached to or adjacenttarget cells.

The two portions of the compound of the invention may be linked togetherby any of the conventional ways of cross-linking polypeptides, such asthose generally described in O'Sullivan et al (1979) Anal. Biochem. 100,100-108. For example, the antibody portion may be enriched with thiolgroups and the enzyme portion reacted with a bifunctional agent capableof reacting with those thiol groups, for example theN-hydroxysuccinimide ester of iodoacetic acid (NHIA) orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioetherbonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimideester, are generally more stable in vivo than disulphide bonds.

It may not be necessary for the whole enzyme to be present in thecompound of the invention but, of course, the catalytic portion must bepresent.

Alternatively, the compound may be produced as a fusion compound byrecombinant DNA techniques whereby a length of DNA comprises respectiveregions encoding the two portions of the compound of the inventioneither adjacent one another or separated by a region encoding a linkerpeptide which does not destroy the desired properties of the compound.Conceivably, the two portions of the compound may overlap wholly orpartly. The antibody component of the fusion must be represented by atleast one binding site. Examples of the construction of antibody (orantibody fragment)-enzyme fusions are disclosed by Neuberger et al(1984) Nature 312, 604.

The DNA is then expressed in a suitable host to produce a polypeptidecomprising the compound of the invention. Thus, the DNA encoding thepolypeptide constituting the compound of the invention may be used inaccordance with known techniques, appropriately modified in view of theteachings contained herein., to construct an expression vector, which isthen used to transform an appropriate host cell for the expression andproduction of the polypeptide of the invention. Such techniques includethose disclosed in U.S. Pat. Nos. 4,440,859 issued 3 Apr. 1984 to Rutteret al, 4,530,901 issued 23 Jul. 1985 to Weissman, 4,582,800 issued 15Apr. 1986 to Crowl, 4,677,063 issued 30 Jun. 1987 to Mark et al,4,678,751 issued 7 Jul. 1987 to Goeddel. 4,704,362 issued 3 Nov. 1987 toItakura et al, 4,710,463 issued 1 Dec. 1987 to Murray, 4,757,006 issued12 Jul. 1988 to Toole, Jr. et al, 4,766,075 issued 23 Aug. 1988 toGoeddel et al and 4,810,648 issued 7 Mar. 1989 to Stalker, all of whichare incorporated herein by reference.

The DNA encoding the polypeptide constituting the compound of theinvention may be joined to a wide variety of other DNA sequences forintroduction into an appropriate host. The companion DNA will dependupon the nature of the host the manner of the introduction of the DNAinto the host, and whether episomal maintenance or integration isdesired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance. Alternatively, the gene for such selectable traitcan be on another vector, which is used to co-transform the desired hostcell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells.

The vectors include a procaryotic replicon, such as the ColE1 ori, forpropagation in a procaryote, even if the vector is to be used forexpression in other, non-procaryotic, cell types. The vectors can alsoinclude an appropriate promoter such as a procaryotic promoter capableof directing the expression (transcription and translation) of the genesin a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

Typical procaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99Aand pKK223-3 available from Pharmacia, Piscataway, N. J., USA.

A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells.

An example of an inducible mammalian expression vector is pMSG, alsoavailable from Pharmacia. This vector uses the glucocorticoid-induciblepromoter of the mouse mammary tumour virus long terminal repeat to driveexpression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markershis3, trp1, leu2 and ura3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3'-single-stranded terminiwith their 3'-5'-exonucleolytic activities, and fill in recessed 3'-endswith their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA encoding the polypeptide of theinvention is to use the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The said specific primers may containrestriction endonuclease recognition sites which can be used for cloninginto expression vectors using methods known in the art.

Exemplary genera of yeast contemplated to be useful in the practice ofthe present invention are Pichia, Saccharomyces, Kluyveromyces, Candida,Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen,Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium,Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred generaare those selected from the group consisting of Pichia, Saccharomyces,Kluyveromyces, Yarrowia and Hansenula. Examples of Saccharomyces areSaccharomyces cerevisiae, Saccharomyces italicus and Saccharomycesrouxii. Examples of Kluyveromyces are Kluyveromyces fragilis andKluyveromyces lactis. Examples of Hansenula are Hansenula polymorpha,Hansenula anomala and HansenuIa capsulata. Yarrowia lipolytica is anexample of a suitable Yarrowia species.

Methods for the transformation of S. cerevisiae are taught generally inEP 251 744, EP 258 067 and WO 90/01063, all of which are incorporatedherein by reference.

Suitable promoters for S. cerevisiae include those associated with thePGK1 gene, GAL1 or GALL10 genes, CYC1, PHO5, TRP1, ADH1, ADH2, the genesfor glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, α-mating factor pheromone,a-mating factor pheromone, the PRB1 promoter, the GUT2 promoter, andhybrid promoters involving hybrids of parts of 5' regulatory regionswith parts of 5' regulatory regions of other promoters or with upstreamactivation sites (eg the promoter of EP-A-258 067).

The transcription termination signal is preferably the 3' flankingsequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3' flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, ie may correspond to the promoter.Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae AHD1 gene is preferred.

According to a further aspect of the present invention, there isprovided a method of destroying target cells in a host, the methodcomprising administering to the host (i) a compound according to thepresent invention, (ii) a cytotoxic agent and (iii) a substance which inits native state is capable of inhibiting the effect of said cytotoxicagent and which can be converted by said inactivating portion into asubstance which has less effect on the cytotoxic agent.

Preferably, the compound of the invention is administered and, oncethere is an optimum balance between the tumour to normal cell ratio ofthe compound and the absolute level of compound associated with thetumour, the cytotoxic agent together with the substance capable ofblocking the effect of the cytotoxic agent are administered. However, analternative method of administration would be possible. The amount ofthe compound of the invention circulating in the blood may be determinedby measuring the activity of the enzymatic portion.

Preferably, the present invention provides a method of treating a mammalharbouring a tumour. Suitably, the mammal is first prepared for tumourtherapy by administering a compound according to the present inventionand allowing the ratio of compound bound to target cells to compound notbound to target cells to reach a desired value. The method then furthercomprises administering to the mammal a cytotoxic agent and a substancewhich in its native state is capable of inhibiting the effect of saidcytotoxic agent from which a substance which has less effect on thecytotoxic agent can be generated by the inactivating portion of the saidcompound.

The compounds of the invention are administered in any suitable way,usually parenterally, for example intravenously, intraperitoneally orintra-vesically, in standard sterile, non-pyrogenic formulations ofdiluents and carriers, for example isotonic saline (when administeredintravenously). The present invention therefore provides a means toallow continuous action of a cytotoxic agent at tumour sites whilstprotecting normal tissues from the effects of the cytotoxic agentexpressed by the target cancer. The substance which in its native stateis capable of inhibiting the effect of the cytotoxic agent is given at adose level sufficient to protect the normal tissues. However, thesubstance reaching tumour sites is inactivated before it can enter thecells and protect them from the cytotoxic agent. In this way, normaltissues are protected from the effects of the cytotoxic agent whereasthe protective molecule is rapidly degraded at tumour sites.

The cytotoxic agent and inhibitor are administered by any of the routesdescribed for the compounds of the invention, and could also beadministered orally.

It has also been shown that antibody-enzyme conjugates reach their peakconcentration in tumours generally within 24 hours and that enzymeactivity persists at tumour sites for up to 7-8 days. Antibody-enzymeconjugates also persist in blood and non-tumour tissues for severaldays. Enzyme in non-tumour tissues will degrade the inhibitor substanceand thus tend to diminish its protective effect or increase therequirement for it. Clearance of enzyme from the blood can beaccelerated or the enzyme can be inactivated without significantlyaltering enzyme levels in tumour sites by several techniques. Thesetechniques may be employed usefully and additionally to the othercomponents.

Although the antibodies or antibody fragments used in theantibody-enzyme conjugate can be `humanised` to reduce theirimmunogenicity (as disclosed by Morrison et al (1984) Proc. Natl. Acad.Sci. USA 81, 6851-6855 and Riechmann et al (1988) Nature 332,323-327),the bacterial enzymes CPG 1 and CPG2 have no human analogue and forrepeated use in man it may be desirable to modify them so as to reducetheir immunogenicity or to employ means to induce immunosuppression orimmune tolerance.

Techniques for reducing the immunogenicity of foreign proteins,applicable to antibody-enzyme conjugates, include that of conjugation toforms of polyethylene glycol (Wilkinson et al (1987) J. Immunol. 139,326-331).

Alternatively, or additionally, the problem of immunogenicity may beovercome by administering immunosuppressors or immune tolerance inducingagents. Cyclosporin and FK506 are widely used drugs to achieveimmunosuppression in tissue transplantation. Cyclosporin has been shownto delay host antibody response to foreign protein (Lederman et al(1988) Br. J. Cancer 58, 562-566 and 654-657). Tolerance to foreignproteins when the host encounters the foreign problem for the first timeafter receiving an antibody directed at the CD4 epitope on lymphocyteshas been disclosed (Waldman et al (1988) pp 16-30 in Progress in Allergy(Shizata & Woksman, Eds, New York). Further means to achieve this havebeen described elsewhere and may change as improvements occur in controlof host antibody responses to foreign antigens. Catalytic antibodies(abzymes) may be `humanized` to reduce or remove their immunogenicity.

When an antibody directed at a tumour associated antigen or anantibody-enzyme conjugate is injected into an appropriate tumour bearinghost, only a small fraction of the antibody or conjugate localises atthe tumour site and most of it remains in blood and other normal tissuesfor several days. Thus, although tumour concentration of the enzyme willbe higher than in normal tissues, the volume of normal tissues is muchgreater. Thus, to minimize the amount of enzyme residual in normaltissues and blood it may be desirable to use the methods of the presentinvention in conjunction with the methods disclosed in WO 89/10140;Bagshawe (1989) Brit. J. Cancer 60, 275-281; and Sharma et al (1990)Brit. J. Cancer 61, 659-662 for inactivating and clearing excessantibody-enzyme conjugate from the blood.

In attempting to achieve eradication of cancers it may not be possibleto avoid suppression of haemopoietic function although for a giveneffect on a tumour target (myelosuppression) it is expected to be muchless with the system described herein. Similarly, for a given degree ofmyelosuppression a much greater tumour effect is expected. Growthfactors acting on haemopoietic tissues may therefore be usefullyemployed in combination with the system described herein.

The system described herein may be used in conjunction with other formsof therapy. These include conventional cytotoxic agents, and use ofmultiple enzyme delivery to inactivate more than one metabolite.

Similarly, an enzyme delivered to tumour sites by an antibody mayfunction both to activate a pro-drug and to inactivate a metabolitewhich protects normal tissues. Carboxypeptidase G2 as disclosed hereininactivates folinic acid at tumour sites to leave the tumour cellsunprotected against trimetrexate. The same tumour located enzyme canactivate a benzoic acid pro-drug to form a cytotoxic mustard (asdisclosed by Bagshawe (1989) Brit. J. Cancer 60 275-281).

Of course, one or more enzymes may be directed to the tumour siteseither by the same or a different antibody, and such that one or moreprodrugs are converted to an active drug, and that one or moreprotective agents are degraded at the tumour site. Thus, two or moreenzymes may be joined to the same antibody.

Since antibody-enzyme conjugates generally reach their maximumconcentration at tumour sites within 12-24 hours and since they may takeseveral days to clear from plasma and other body fluids, it has alsobeen shown that it is advantageous to accelerate the clearance ofantibody-enzyme conjugate and to inactivate the specific enzyme presentin the blood. Several means by which this may be achieved have beendescribed (WO 89/10140).

The antibody used for clearing or inactivating the antibody-enzymeconjugate can be directed towards the antigen binding site on theantitumour antibody, or the active site of the enzyme, or any other siteon the antibody-enzyme conjugate. Such antibodies may have additionalgalactose residues or other sugars added to accelerate clearance or maybe desialylated. Galactosylation of the antibody results in its rapidclearance from the blood through take-up by galactose receptors onhepatocytes. Alternatively, or additionally, the antibody-enzymeconjugate is galactosylated, and given after the hepatic galactosereceptors have been blocked by asialo-bovine submaxillary glandmucoprotein or antibody directed at hepatic galactose receptor or othermolecule with high affinity for galactose receptor. The blockingsubstance is maintained in plasma for a period of up to 24 hours so thatthe antibody-enzyme complex localises at tumour sites but followingcessation of galactose receptor blockade, the galactosylatedantibody-enzyme is quickly cleared via the available galactosereceptors.

The present invention will now be illustrated by way of the followingExamples and Figures with specific references to cytotoxic therapy withtrimetrexate and folinic acid rescue.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structures of trimetrexate, methotrexate, folinic acid,CB3717 and ICI D1694.

FIG. 2 is a diagrammatic representation of the invention wherein thetarget-cell specific portion is an antibody and is coupled tocarboxypeptidase G2; folinic acid is the inhibitor of a cytotoxic agent;and trimetrexate is the said cytotoxic agent.

EXAMPLE 1

Cytotoxic therapy with trimetrexate and folinic acid rescue

The present invention may provide a means to allow more continuousanti-folate action at tumour sites whilst protecting normal tissues fromanti-folate effects. The first step is to initiate immunosuppressive orimmune tolerance inducing agents and this will normally occur not lessthan 2 days before the compound of the present invention isadministered. If the antibody-enzyme conjugate is non-immunogenic thisstep is omitted. An antibody-CPG2 conjugate, or antibody fragmentconjugated chemically or by recombinant DNA technology to CPG2 orsimilar enzyme is given by intravenous or other appropriate route; theantibody is directed at a tumour antigen expressed by the target cancer.After several hours has been allowed for the conjugate to localise attumour sites a second antibody is given. This may be directed at theactive site on the enzyme in which case additional galactose residuesare attached to the second antibody to ensure its rapid clearance viahepatocyte galactose receptors. Alternative mechanisms for rapidclearance of the conjugate from non-tumour sites have been described.When enzyme levels have fallen to very low or undetectable levels inplasma, trimetrexate is given by bolus or by continuous infusions withthe aim of maintaining a constant plasma concentration. Concurrentlywith the trimetrexate, folinic acid is given by intravenous infusion ata dose level sufficient to protect the normal tissues from trimetrexatetoxicity. Folinic acid reaching tumour sites where CPG2 is located isdeglutamated as is folio acid and rendered inactive before it can entercells and protect them from trimetrexate. In this way normal tissues maybe protected by folinic acid from the action of trimetrexate whereas theprotective molecule is rapidly degraded at tumour sites. Otherantifolate drugs which, like trimetrexate are not degraded by CPG2, maybe substituted for trimetrexate.

Since the trimetrexate blockade of dihydrofolate reductase may be, atleast partially, bypassed by endogenous sources of thymidine, athymidine-degrading enzyme might also be targeted to tumour sites byantibody or other ligand.

It is recognised that distribution of the antibody-enzyme conjugates inthe tumour will not be uniform since there is heterogeneity in theexpression of tumour-associated antigens on cell membranes. Secretion ofan antigen into extracellular space results in more uniform distributionof target antigen and secreted antigen forms complexes with targetedantibody-enzyme conjugate. Internalisation of the antibody-enzymeconjugate by tumour cells is not necessary and may be undesirable.

It is evident that the method according to the present invention differsfrom conventional cytotoxic chemotherapy with methotrexate and folinicacid rescue by the introduction of a means of degrading the normalmetabolite (folic acid) and the rescue agent (folinic acid) but not thefolic acid antagonist at tumour sites.

Loss of enzyme from tumour sites by dissociation of the antibody fromtarget antigen and by biodegradation are likely to result after somedays in reduced levels of enzyme at those sites. The rate of fall inenzyme activity will vary with tumour and enzyme type; carboxypeptidaseactivity has been detected in human colonic carcinoma xenograftedtumours in nude mice 7 days after administration conjugated to anantibody directed at CEA. It should therefore be possible to continuetrimetrexate therapy for about 5 days with an interruption about 24-48hours, during which time further antibody-enzyme conjugate could begiven and cleared from non-tumour sites with further cycles to followprovided there is effective immunological control or avoidance of hostantibody response.

Thus although the proposal would not result in uninterrupted therapy theratio of treatment period to treatment free period in conventionaltherapy of about 1:7 would be improved to about 2-3:1.

EXAMPLE 2

Cytotoxic therapy with thymidylate synthetase inhibitors and thymidinerescue

The same principle may be applied to other anti-metabolite therapies.For instance, considerable effort has been directed in recent years tothe development of agents that block the key enzyme thymidylatesynthetase such as CB3717 and ICI D1694 (Jodrell et al 1991. BJC 64,833-8). Such agents are likely to suffer the same limitations asconventional cytotoxic agents in that they also block thymidylatesynthetase in normal cells. Their action is reversible by thymidine.Thymidine administration may therefore be used to protect normal tissuesfrom thymidylate synthetase inhibitors whilst the protective effect ofthe thymidine and endogenous thymidine may be limited at tumour sites bya thymidine degrading enzyme, such as dihydrothymine dehydrogenase, orthymidine kinase which would inhibit entry of thymidine into the tumourcells. The thymidine inhibitor enzyme or agent is delivered by antibodyor other appropriate vector.

Dihydrothymine dehydrogenase can be obtained from normal humanlymphocytes (Shiotani & Weber 1989 Cancer Res. 49, 1090-1094) andtherefore has the advantage that an antibody-enzyme conjugate of lowimmunogenicity may be produced by its conjugation to a human or`humanised` antibody.

It is also envisaged that both an antifolate agent and an anti-thymidineagent could be used together with the appropriate inhibitors andinhibitor degrading enzymes resulting in an effective cytotoxiccombination.

EXAMPLE 3

Combination therapy with ADEPT

The ADEPT concept uses an antibody-enzyme conjugate to generate acytotoxic drug from an inactive precursor at tumour sites. The presentinvention can be used in conjunction with ADEPT thus resulting in a formof combination chemotherapy, both active drugs being confined largely tothe tumour site but employing the same, or different, enzymes to producethe active agent in one case and to destroy a protective agent in theother.

In the case of ADEPT treatment, nude mice bearing human choriocarcinoma(CC3) tumours received 29 units of CPG2 conjugated to anti-HCG (W 14Fab₂, as disclosed in WO 88/07378), and after 24 or 48 hours receivedpro-drug (41 μM/kg). The amount of protective agent is adjusted to givean optimal protective effect in the absence of inhibitory agent.

EXAMPLE 4

Treatment of patient with hepatic metastases and abdominaladenocarcinoma

Starting on day minus-2 a patient with large hepatic metastases andrecurrent abdominal adenocarcinoma of the colon received theimmunosuppressive agent cyclosporin by continuous intravenous infusion.On day 0 he received a compound containing the F(ab')₂ portion of amonoclonal antibody known as A₅ B₇ which binds carcinoembryonic antigen(CEA) chemically conjugated to carboxypeptidase G2 a bacterial enzyme.The amount of enzyme given was 20000 enzyme units (10,000 U/m²) byintravenous infusion over 2 hours. 24 hours later (day 1) the enzyme wasstill available in his blood plasma at 1.0 unit/ml and he then received80 mg (40 mg/m²) of monoclonal antibody SB43 which had beengalactosylated to accelerate its clearance. This was administered over24 hours by continuous intravenous infusion and during the last hour ofthe infusion 10 mg of non-galactosylated SB43 was added to the infusate.Following completion of the SB43 infusion (day 2) when CPG2 activity wasno longer detectable in plasma, trimetrexate was given in 5% dextrose byintravenous bolus. Infusion of folinic acid 100 mg commenced at thistime by continuous 24 hour infusion. Trimetrexate was continued dailyfor 4 days (day 2, 3, 4, 5).

The cycle starting with the antibody enzyme conjugate restarted on days7 and 14. Toxicity was confined to a temporary rise in serum creatine, awell-known complication of therapy with cyclosporin. The host antibodyresponse to the tumour antibodies and bacterial enzymes was notdetectable until after completion of the three cycles of treatment.

The peripheral blood white cell and platelet counts remained at normalor supranormal levels during and after this treatment. Subsequentpatients receive escalating doses of trimetrexate or reduced doses offolinic acid.

The monoclonal antibody A₅ B7, raised in mice to carcinoembryonicantigen (CEA), is available from Cancer Research Campaign Technology,Cambridge House, 5-10 Cambridge Terrace, Regent's Park, London NW1 4JL,and was prepared by the method disclosed by Harwood et al (1986) Brit.J. Cancer 54, 75-82 and Example 5. The murine monoclonal antibody SB43is available from Cancer Research Campaign Technology and was preparedby the method disclosed by Sharma et al (1990) Brit. J. Cancer 61,659-662 and Example 8.

EXAMPLE 5

Preparation of a monoclonal antibody reactive against carcinoembryonicantigen

Purified CEA was prepared from metastases of colonic tumour.Radio-iodination to a specific activity of 6 μCiμg⁻¹ was carried out bythe iodogen method. Dilution buffer was prepared as a 0.15M sodiumphosphate buffer, pH 7.4, containing 0.1% bovine serum albumin. Thestudies at low ionic strength were carried out in 0.02M Tris-HCl bufferat pH 7.4.

Immunisation schedule: Monoclonal antibody A₅ B₇ was raised againstpurified, heat-treated CEA using the following procedure. One milligramof purified CEA was heated at 85° C. for 35 min in 0.05M phosphatebuffer (pH 7) at a concentration of 1 mg ml⁻¹. After mixing with 1 ml of10% aqueous potassium aluminium sulphate (alum), the pH was adjustedwith constant stirring to 6.5-7 by dropwise addition of NaOH solution.After stirring at room temperature for 30 min the resulting precipitatewas washed three times in saline. It was then mixed with 10¹⁰ formalisedBordetella pertussis (kindly supplied by Wellcome ResearchLaboratories). Three different immunisation schedules were used.

Spleen cells from the immunised mice were then fused with eitherSP2/0-Ag 14 or P3-NS/1-Ag 4-1 myeloma cells (Flow Laboratories, UK) andthe hybridomas producing anti-CEA cloned by single cell transfer.

EXAMPLE 6

Preparation of F(ab')₂ fragments of A₅ B₇

The monoclonal anti-CEA (A₅ B₇) used in this study has been describedpreviously and chosen for its low cross-reactivity with NCA and itsstability on immunopurification and radiolabelling. F(ab')₂ fragmentswere prepared by the method Lamoyi and Nisonoff (1983) J. Immunol.Methods 56, 235-243. After separation of the digest mixture on SephacrylS-200, the fractions were analysed by SDS-PAGE using a 7.5% gel. Thefraction containing the F(ab')₂ was concentrated and dialysed against0.15M phosphate buffer, pH 7. Both intact A₅ B₇ and the fragment wereshown to be immunologically active and relatively homogeneous byelectroblotting of the SDS gel onto nitrocellulose paper and overlayingwith ¹²⁵ I-labelled CEA. Intact A₅ B₇ and its F(ab')₂ fragment wereradiolabelled by the chloramine T method to specific activities of 5.6and 5.2 μCi/μg respectively. Both labelled preparations were shown toretain immunological activity by solid-phase radioimmunoassay using CEAcoupled to amino-cellulose (Rogers et al (1983) Eur. J. Cancer Clin.Oncol. 19, 629-639). An excess of 60% activity was retained in eachcase.

EXAMPLE 7

Conjugation of A₅ B₇ -F(ab')₂ to CPG2

To a solution of CPG2 (2.15 mg) in phosphate buffer (2.5 ml, pH=7.6,containing 1 mM EDTA) was added 4-(p-maleimidophenyl) butyric acidN-hydroxysuccinimide ester (138 μg) in DMF (13.5 μl) corresponding to a15 molar excess of ester over carboxypeptidase. The solution was leftfor 2 hours after which time excess ester was removed by gel filtration(PD10 column), eluted with 3.2 cm phosphate/EDTA buffer)--Solution 1.

F(ab')₂ -A₅ B₇ (2.6 rag) in phosphate/EDTA buffer (1.2 ml) was treatedwith S-acetyl thioglycol acid-N-hydroxysuccinimide ester (SATA) (90 μg)in DMF (10 μl) and the mixture left for 1 hour at room temperature.Excess SATA was removed by gel filtration (PD10 column, eluted with 3.2cm phosphate buffer).

Thirty minutes before use, a solution of NH₄ OH (3.47 g neutralised withNa₃ PO₄ H and NaOH solution and made up to 100 ml with H₂ O containing930 mg EDTA) (320 μl) was added--Solution 2.

The solutions 1 and 2 were then mixed and allowed to stand at 4° C.overnight. After concentration (ten-fold) (Minicon) the conjugate wasisolated by gel filtration (S-12 column) on FPLC (Pharmacia). Theconjugate was filtered through a Millipore 0.22 μm filter. The proteinconcentration was measured and the enzyme activity measured onspectrophotometric assay before use.

EXAMPLE 8

Production of a monoclonal antibody reactive against CPG2

A monoclonal antibody (SB43) raised against CPG2 is used for makingbispecific antibody (see next Example) and for clearance andinactivation of residual enzyme activity at non-tumour sites.

The monoclonal antibody was made in the following way. Balb/C mice (6-8weeks old) were immunised with 50 μg CPG2 i.p. in incomplete Freund'sadjuvant followed by two injections of CPG2 in complete Freund'sadjuvant (50 μg CPG2 each, i.p.) at monthly intervals and with two dailyinjections (50 μg and 100 μg in PBS, i.v.) 2 days before fusion. Immunespleen cells were fused with non-immunoglobulin secreting SP2/0 myelomacells according to the hybridoma procedures of Kohler and Milstein(1975) Nature 256, 495.

The presence of anti-CPG2 antibodies was detected by a solid-phaseindirect radioimmunoassay. A 1 μg ml⁻¹ solution of CPG2 in 0.05Mphosphate buffer was placed in polyvinyl microtitre plates (100 ng perwell), allowed to dry, fixed with methanol and washed with PBS buffercontaining a 0.05% Tween and 0.1% bovine serum albumin. Supernatant orpurified antibody samples were diluted in PBS and incubated in the CPG2coated microtitre plates (100 μl per well) at 37° C. for 4 h and thenfor 1 h with ¹²⁵ I-labelled rabbit anti-mouse IgG. The wells were washedthree times with PBS-Tween buffer between each stage and after finalwashing individual wells were cut and counted in a gamma counter.

EXAMPLE 9

Bispecific antibody reactive against CPG2 and CEA The hybridomaproducing A₅ B7, a monoclonal antibody reactive against CEA has beendisclosed by Harwood et al (1986) Brit. J. Cancer 54, 75-82, and amethod of generating a hybridoma producing SB43, a monoclonal antibodyreactive against CPG2 is disclosed in the Examples.

The fusion protocol allows any two antibody-producing hybridomas to befused and has been disclosed previously (Clark & Waldmann (1987) J.Natl. Cancer Inst. 79, 1393-1401). Briefly, 5×10⁶ to 3.5×10⁷ cells ofone parental hybridoma that have been previously renderedhypoxanthine/aminopterin/thymidine (HAT) sensitive by selection for ahypoxanthine phosphoribosyltransferase-negative variant were fused at 11:1 or 10:1 ratio, using 1 ml of a 50% (wt/vol) solution of polyethyleneglycol, with the second parental hybridoma cells that had beenpretreated with a lethal dose of 10 mM iodoacetamide. Excesspolyethylene glycol was washed out and the cells were plated atconcentrations from 8×10⁵ per ml to 2×10⁵ per ml into 24-well plates inbicarbonate-buffered Iscove's modified Dulbecco's medium (IMDM)supplemented with 5% (vol/vol) fetal calf serum. After 24 hr in culture,hybrid hybridomas were selected for in medium containing HAT.

EXAMPLE 10

Reduction of residual enzyme activity at non-tumour sites

It is desirable to inactivate the enzymatic portion of theenzyme-antibody conjugate at non-tumour sites, but not at the tumour.One method of achieving this effect is to administer to the patientbeing treated using the compounds of the invention antibodies raisedagainst the enzyme portion which have been conjugated with galactoseresidues.

A monoclonal antibody (SB43) directed at CPG2 inactivates the enzyme. Toprevent the antibody inactivating the enzymes at tumour sites additionalgalactose residues are conjugated to it so that it can still inactivateenzyme in plasma when it is given by intravenous route but theinactivating antibody is rapidly removed from the plasma and galactosereceptors on hepatocytes.

The galactosylated SB43 is given to eliminate enzymatic activity inplasma and then to give an amount of the non-galactosylated SB43 toinactivate residual enzyme activity in other non-tissues.

The monoclonal antibody SB43 is galactosylated using the followingprotocol: A stock solution of the activated derivative was made up asfollows: Cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside(Sigma C-4141) [400 mg] in anhydrous methanol (10 ml) was treated with5.4 mg of sodium methoxide in 1 ml of anhydrous methanol at roomtemperature for 48 hours. The mixture was kept in a 25 ml Quickfit(Trademark) conical flask fitted with a slightly greased stopper.

A stock solution of SB43 (1.3 mg/ml) was prepared in 0.25M sodium boratebuffer, pH 8.5. Aliquots of the required amount of activated galactosylderivative (80, 40, 20, 10 μl ) were dispensed into 3 ml glass ampoulesand evaporated to a glassy residue in a stream of nitrogen. A solutionof the antibody (200 μg) was added mixed until the residue wasdissolved. After 2 hours at room temperature, the solution was dialysedagainst three changes of PBS.

The preparations were scaled up by taking multiples of the volumesmentioned above.

I claim:
 1. A cancer therapeutic system comprising:(i) a cytotoxic cancer therapeutic agent that inhibits enzymatic activity; (ii) an inhibitor of said cytotoxic cancer therapeutic agent, wherein said inhibitor, in its native state, is capable of inhibiting the cancer cell killing effect of said cytotoxic cancer therapeutic agent; and (iii) a targeting and inactivating compound, said compound comprising:a cancer cell specific binding portion that is a monospecific antibody or an antigen binding fragment thereof; a bispecific antibody; or a ligand which specifically binds to a cell surface receptor characteristic of a cancer cell; and an inactivating portion which converts said inhibitor so that said inhibitor has less inhibitory effect on the cytotoxic cancer therapeutic agent, said inactivating portion selected from the group consisting of: an enzyme and an abzyme; wherein said cancer cell specific binding portion is coupled to said inactivating portion.
 2. A method of destroying target cells in a host, the method comprising administering to the host(i) a cytotoxic cancer therapeutic agent that inhibits enzymatic activity; (ii) an inhibitor of said cytotoxic cancer therapeutic agent, wherein said inhibitor, in its native state, is capable of inhibiting the cancer cell killing effect of said cytotoxic cancer therapeutic agent; and (iii) a targeting and inactivating compound, said compound comprising;a cancer cell specific binding portion that is a monospecific antibody or an antigen binding fragment thereof; a bispecific antibody; or a ligand which specifically binds to a cell surface receptor characteristic of a cancer cell; and an inactivating portion which converts said inhibitor so that said inhibitor has less inhibitory effect on the cytotoxic cancer therapeutic agent, said inactivating portion selected from the group consisting of: an enzyme and an abzyme; wherein said cancer cell specific binding portion is coupled to said inactivating portion.
 3. A method of treating a mammal harbouring a tumour, the mammal having been prepared for treatment by administering a targeting and inactivating compound and allowing the compound bound to cancer cells to compound not bound to cancer cells ratio to reach a desired value, said compound comprising:a cancer cell specific binding portion that is a monospecific antibody or an antigen binding fragment thereof; a bispecific antibody; or a ligand which specifically binds to a cell surface receptor characteristic of a cancer cell; and an inactivating portion which converts an inhibitor so that said inhibitor has less inhibitory effect on a cytotoxic cancer therapeutic agent that inhibits enzymatic activity, said inactivating portion selected from the group consisting of: an enzyme and an abzyme; wherein said cancer cell specific binding portion is coupled to said inactivating portion;the method comprising administering to the mammal said cytotoxic cancer therapeutic agent that inhibits enzymatic activity and said inhibitor of said cytotoxic cancer therapeutic agent, wherein said inactivating portion bound to cancer cells converts said inhibitor so that said inhibitor has less inhibitory effect on the cytotoxic cancer therapeutic agent.
 4. A cancer therapeutic system according to claim 1 wherein the cytotoxic cancer therapeutic agent is a folic acid antagonist and the inhibitor of said cytotoxic cancer therapeutic agent is folinic acid or an analogue thereof.
 5. A cancer therapeutic system according to claim 1 wherein the inactivating portion of said targeting and inactivating compound is an enzyme.
 6. A cancer therapeutic system according to claim 1 wherein the cancer cell specific binding portion is a monospecific antibody or an antigen binding fragment thereof.
 7. A cancer therapeutic system according to claim 6 wherein the monospecific antibody or antigen binding fragment thereof binds to a tumour-associated antigen.
 8. A cancer therapeutic system according to claim 1 wherein the cancer cell specific binding portion is a ligand which specifically binds to a cell surface receptor characteristic of a cancer cell.
 9. A cancer therapeutic system according to claim 1 wherein the inhibitor is folinic acid or an analogue thereof which is capable of inhibiting the cancer cell killing effect of a cytotoxic cancer therapeutic agent which acts on the enzyme dihydrofolate reductase.
 10. A cancer therapeutic system according to claim 9 wherein the inactivating portion comprises at least the catalytic portion of carboxypeptidase G2.
 11. A method according to claim 2 or 3 wherein the inactivating portion of said targeting and inactivating compound is an enzyme.
 12. A method according to claim 2 or 3 wherein the cancer cell specific binding portion comprises a monospecific antibody or an antigen binding fragment thereof.
 13. A method according to claim 12 wherein the monospecific antibody or antigen binding fragment thereof binds to a tumour-associated antigen.
 14. A method according to claim 2 or 3 wherein the cancer cell specific binding portion is a ligand which specifically binds to a cell surface receptor characteristic of a cancer cell.
 15. A method according to claim 2 or 3 wherein the inhibitor is folinic acid or an analogue thereof which is capable of inhibiting the cancer cell killing effect of a cytotoxic cancer therapeutic agent which acts on the enzyme dihydrofolate reductase.
 16. A method according to claim 15 wherein the inactivating portion comprises at least the catalytic portion of carboxypeptidase G2.
 17. A cancer therapeutic system according to claim 1 wherein the cytotoxic cancer therapeutic agent is a thymidine antagonist and the inhibitor of said cytotoxic cancer therapeutic agent is thymidine or an analogue thereof. 